2.1.

Animals

All mice were either wild type or transgenic males on the 129SvEv/C57BL/6N F1 hybrid background. The experiments were limited to the male mice, to avoid potential sex-dependent LTP variability, which has been reported both in the amygdala^23,24 and hippocampus.^25,26 To obtain the mice-expressing hM4Di²⁷ in Sst-INs or PV-INs, homozygous R26-LSL-Gi-DREADD males (JAX Stock No: 026219) on C57BL/6N background were crossed with homozygous interneuron-specific Cre driver females on 129SvEv background—either the Sst-IN-specific Cre driver (Sst-Cre), $Ss{t}^{tm2.1(cre)Zjh}$²⁸ (JAX: 013044) or the PV-IN-specific Cre driver (PV-Cre), $Pval{b}^{tm1(cre)Arbr}$²⁹ (JAX: 008069). The expression specificity of floxed reporters in the BLA of these driver lines has been validated using immunostaining and whole-cell recordings by the Luthi lab.²² To obtain heterozygous Sst-Cre mice, wild type C57BL/6N males were crossed with the 129SvEv homozygous $Ss{t}^{tm2.1(cre)Zjh}$ females. All breeding were the trios of one male and two females on the C57BL/6N and 129SvEv backgrounds, respectively. Male pups were weaned at p21–p25 and housed three to five littermates per cage. All experiments were approved by Virginia Tech IACUC and followed the NIH Guide for the Care and Use of Laboratory Animals.

2.2.

Surgery—Viral Injection and Optrode Implantation

Adenoassociated viruses (AAVs) for expressing Chronos or Cre-activated Arch were generated from pAAV-Syn-Chronos-GFP³⁰ (Addgene #59170) or pAAV-FLEX-Arch-GFP (Addgene #22222), respectively, gifts from Edward Boyden. The viruses (pseudotype 5 for Chronos and pseudotype 1 for Arch) were prepared by the University of North Carolina Vector Core (Chapel Hill, North Carolina). At p28, the heterozygous Sst-Cre male mice were anesthetized by intramuscular injection of ketamine/xylazine/acepromazine, $100/5.4/1\mathrm{mg}/\mathrm{kg}$, as routinely done for electrode implantations in the Buzsaki lab,³¹ in the volume not exceeding 0.05 ml, placed in a stereotaxic apparatus (David Kopf, Tujunga, California) and underwent minimum craniotomy ($\sim 0.5\mathrm{mm}$ diameter). For the dmPFC virus injection, the dura mater was preserved. A heater-pulled short-taper glass pipette (shaft: $0.6/0.4\mathrm{mm}$ external/internal diameter, beveled tip: $50\mu \mathrm{m}$, diameter, Drummond, Broomall, Pennsylvania) filled with the virus solution (${10}^{12}\text{viral particles}/\mathrm{ml}$) was slowly lowered to the target (1.3 mm anterior, 0.4 mm lateral from bregma, and 1.3 mm ventral from brain surface). The solution ($0.5\mu \mathrm{l}$) was injected bilaterally at the rate of $0.2\mu \mathrm{l}/\min$ using a syringe pump connected to the pipette through plastic tubing filled with water as described.³² For the BLA virus injections, the dura mater was removed to allow straight penetration by a less rigid long-taper pipette. The virus solution ($0.4\mu \mathrm{l}$, ${10}^{12}\text{particles}/\mathrm{ml}$) was injected bilaterally at the rate of $0.1\mu \mathrm{l}/\min$ at the coordinates (1.2 mm posterior, 3.2 mm lateral from bregma, and 4.2 mm ventral from brain surface). Custom-made optrodes were fabricated with a miniature LED [C460EZ500, CREE, peak wavelength 463.3 nm, bandwidth 20 nm full-width half maximum (FWHM)], coupled to an optical fiber (0.66 NA, 0.2-mm core diameter, Prizmatix) with UV-cured index-matched glue (Norland Optical Adhesive 85, Norland) and two $33\text{-}\mu \mathrm{m}$ tungsten wires (polyimide-insulated, California Fine Wire) extended 0.5 mm beyond the optical fiber. The optrodes for in vivo recording were implanted in BLA at p60, as described.³³ For postoperation analgesia, ketoprofen ($5\mathrm{mg}/\mathrm{kg}$) was administered subcutaneously.

2.3.

Ex Vivo Recordings

2.4.

In Vivo Recordings

The subject animals, bilaterally injected with the AAV-Chronos in the dmPFC, AAV-Arch in BLA, and bilaterally implanted with the optrodes in the BLA, were housed with the littermates until the experiment. Using the RHA2000-Series Amplifier USB Evaluation Board (RHA2000-EVAL, Intan Technologies), the local field potentials (LFPs) were recorded from BLA of the subject mouse in the home cage without the lid, from where the cagemates were removed temporarily for the duration of the recording. Mice were habituated to the recording environment by connecting to the recording system for 2 to 3 h per day during 2 to 3 consecutive days. Field excitatory postsynaptic potentials (fEPSPs) were elicited in BLA by blue light stimulation of dmPFC terminals expressing Chronos. The strength of the test pulses (1 ms, 2 to 3 mW at the tip of optrode) was adjusted to obtain the fEPSP slope at 30% to 40% of the maximum. The LED driver (PlexBright LD-1, Plexon) was analog-modulated by DAQ (Analog Shield, Digilent). The LED driving current was routed to the optrodes in either hemisphere by electrical relays (Arduino 4 relays shield, Arduino). Arduino with a custom Arduino sketch controlled both the DAQ and the relays to give the light stimulation on each side every 30 s, alternating the sides every 15 s. Once the baseline of evoked fEPSPs stabilized, LTP was induced by the same blue light stimulation protocol as in the LFP and LTP experiments ex vivo [Fig. 3(a)], except that the protocol was repeated three times for every 1 h. The positions of the optrodes were confirmed by histological analysis.

2.5.

Immunofluorescence

Mice were anesthetized with an intraperitoneal injection of avertin, $0.4\mathrm{mg}/\mathrm{kg}$, and intracardially perfused with paraformaldehyde (PFA, 4%). Brains were postfixed in PFA overnight and sliced into $75\mu \mathrm{m}$ sections. The slices were stained using a rat monoclonal Sst antibody MAB354 (Millipore) dilution 1:600, followed by a Cy3 conjugated donkey antirat antibody (1:400) and imaged using Zeiss LSM710 confocal microscope.

2.6.

Data Analysis

Data were processed using custom scripts written in MATLAB (MathWorks) and Clampfit software (Molecular Devices). Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, La Jolla, California). Normality was tested using the Shapiro–Wilk test. Datasets with normal distribution were compared using the one-sample $t$-test. The datasets with non-normal distribution were analyzed using the Mann–Whitney test and the Wilcoxon’s signed rank test. The difference was deemed significant with $p<0.05$.

3.1.

DREADD-hM4D(Gi) Suppresses GABA Release from BLA Interneurons

The efficiency of DREADD suppression was tested by double-patch recording from connected pairs of an IN-expressing hM4Di-Citrine identified by the fluorescence and a putative principal neuron (PN). The brief depolarizing current was injected in the IN to trigger single AP. It resulted in the inhibitory postsynaptic current (IPSC) in the connected PN, including clozapine-N-oxide (CNO) ($1\mu \mathrm{M}$) in the bath did not prevent APs but diminished IPSCs (Fig. 1). The DREADD suppression of presynaptic GABA release despite the presence of AP was consistent with published findings.³⁷

Fig. 1

DREADD suppresses GABA release from BLA INs during APs. (a) Example of the paired whole-cell recording from a BLA slice. (a) Left: Infrared (IR) image at low magnification. Right: high magnification IR and fluorescent images. Dotted yellow and black circles indicate an Sst-IN expressing hM4D(Gi)-citrine identified by fluorescence and a putative PN, respectively. (b) Examples of double patch recordings of APs evoked by current injection in the INs (400 pA, every 15 s) (upper) and of the corresponding IPSCs in the connected PNs (lower), in the absence of CNO (no CNO, blue) and after 10 min perfusion with $1\mu \mathrm{M}$ CNO (CNO, red). Fifteen traces and IPSC averages (red line) are shown for a pair with an Sst-IN [left, Sst-hM4(Di)] and a pair with a PV-IN [right, PV-hM4(Di)]. (c) Summary data for IPSC amplitudes ($n=4$, including three pairs with Sst-INs, data point connected with continuous lines, and one pair with PV-IN, data points connected with dashed line) in the absence and presence of CNO. *$p<0.05$, Mann–Whitney test.

3.2.

Chemogenetic Suppression of Sst-INs Enables LTP Induction Ex Vivo

For faithful activation of dmPFC axons at high frequency, a fast opsin Chronos³⁰ was expressed in dmPFC. The 50-Hz trains of light pulses were used for LTP induction [Fig. 2(a)]. First, we examined the effect of DREADD suppression of Sst-INs on LTP by whole-cell recording from PNs in BLA slices expressing hM4Di in Sst-INs. In the absence of CNO, the 50-Hz stimulation of dmPFC axons caused a brief post-tetanic potentiation of the excitatory postsynaptic currents (EPSCs), followed by a rapid EPSC decline with a tendency toward depression at the 25 to 30 min after the induction ($p=0.068$, one-sample $t$-test). In the presence of CNO, the stimulation caused increases in EPSCs lasting for at least 30 min (Fig. 2), suggesting that suppression of Sst-INs enables LTP induction.

Repeated neuronal stimulation over extended time intervals, or the spaced LTP protocols, induces LTP more effectively than the shorter protocols, which is consistent with greater efficiency of spaced over massed training.³⁸ However, our attempts to induce LTP with a spaced protocol during the whole-cell recording have failed (data not shown), presumably because the dialysis of the intracellular content limits the time between obtaining the whole-cell configuration and effective LTP induction. To overcome this limitation, in the following experiments, LTP was tested by recording LFPs and using the spaced LTP protocol [Fig. 3(a)]. We run the CNO control and then tested the effects of suppressing PV-INs, and re-examined the effect of suppressing Sst-INs.

Fig. 2

DREADD suppression of Sst-INs enables the facilitation of EPSCs in dmPFC-BLA pathway. (a) LTP induction protocol and (b) relative EPSC amplitudes. Symbols (black triangles: no CNO, red diamonds: CNO) represent the average amplitudes of two consecutive EPSCs recorded during each minute. Upper insets: examples of averaged EPSCs before (1) and after (2) LTP induction as indicated by horizontal gray and black bars, respectively. Scales: 100 pA, 50 ms. (c) Relative EPSC amplitudes averaged during (2) for each neuron. $n=10\text{cells}/\text{slices}$ from six mice (no CNO) and nine cells/slices from five mice (CNO). **$p<0.01$, compared to 1, which is the averaged relative EPSC amplitude during the baseline indicated by gray bars (1), one-sample $t$-test. Error bars on panel (b) represent standard error of the mean (SEM). Boxes and the thick bars inside on panel (c) represent SEM and means.

Fig. 3

DREADD suppression of Sst-INs enables facilitation of LFPs evoked in the dmPFC-BLA pathway. (a) LTP induction protocol. (b–d) LTP experiments on slices without hM4Di (b), with hM4Di expressed in PV-INs (c), and with hM4Di expressed in Sst-INs (d). Left: relative fEPSP slopes. Symbols on the diagram represent the averages of three consecutive data points obtained every 20 s. Insets represent examples of averaged fEPSPs before (1) and after (2) LTP induction, as indicated by horizontal gray and black bars. Scales: 0.1 mV, 10 ms. Right: relative fEPSP slopes averaged during (2) for each slice. $n=16$ slices from four mice (no CNO) and 14 slices from five mice (CNO) in (b). $n=12$ slices from four mice (no CNO) and 11 slices from four mice (CNO) in (c). $n=11$ slices from three mice (no CNO) and 15 slices from six mice (CNO) in (d). ****$p<0.0001$, compared to 1, which is the averaged relative fEPSP slope during the baseline indicated by gray bars (1), one-sample $t$-test. Boxes and the thick bars inside represent SEM and means.

For CNO control, we recorded from slices that did not express hM4Di. There was no significant LTP in the absence or presence of CNO, but there was a tendency toward LTP with CNO [$p=0.09$, one-sample $t$-test, Fig. 3(b)]. In slices with hM4Di in PV-INs, there was no significant LTP in the absence or presence of CNO but a tendency toward LTP in the absence of CNO [$p=0.09$, one-sample $t$-test, Fig. 3(c)]. In slices expressing hM4Di in Sst-INs, there was a significant LTP in the presence of CNO and no LTP in the absence of CNO [Fig. 3(d)]. These data indicate that (a) CNO in the absence of hM4Di has a minor effect if any on LTP induction, (b) suppression of PV-INs does not aid LTP induction but may rather impede it, and (c) suppression of Sst-INs enables LTP.

3.3.

Arch in Sst-INs Enables LTP Induction Ex Vivo and In Vivo

To examine the effects of optogenetic suppression of Sst-INs, Arch-GFP was expressed in the Sst-neurons of BLA [Figs. 4(a) and 4(b)]. Immunostaining revealed that $82\pm 18\%$ of Arch-GFP expressing neurons were costained with the anti-Sst antibody, and $65\pm 11\%$ of Sst-positive neurons expressed Arch-GFP ($n=8$ BLA slices from two mice). Presynaptic stimulation was given through Chronos expressed in dmPFC terminals in the same way as in the DREADD suppression experiments. Paired recordings confirmed that Arch attenuated GABAergic transmission between Sst-IN and PN in BLA ( Supplemental Fig. S1).

Fig. 4

Arch suppression of Sst-INs enables LTP induction. (a) Arch-GFP is expressed mostly in Sst-INs of BLA of an Sst-Cre driver mouse transduced with the floxed Arch AAV in BLA. Confocal image of the somatostatin immunostaining (right panel, Sst, red), Arch-GFP fluorescence (center, GFP, green) and the merged image (left, GFP + Sst). Yellow arrows indicate coexpression of Arch-GFP and Sst, red—Sst only, blue—Arch-GFP only. (b) Examples of slices used for recording. Upper: BLA slice from an Sst-Cre driver mouse transduced with Chronos-AAV in dmPFC and floxed Arch AAV in BLA. Lower: the slice from a mouse transduced only with Chronos-AAV in dmPFC. Fluorescent (GFP) and IR images of the same slices are shown. (c) Left: slice cutting plane. Right: a visible light image of a fixed $150\text{-}\mu \mathrm{m}$ BLA slice cut under the same angle as the $300\mu \mathrm{m}$ “live” slices used for recording. (d) LTP induction protocol. (e) LTP experiments on slices with Arch (left) and without Arch (middle) in Sst-INs, with LTP induced using pulses of blue light alone (black circle: no yellow) or combined with the continuous yellow light of two intensities: $0.15{\mathrm{mW}/\mathrm{mm}}^2$ (orange circles: low yellow) and $0.24{\mathrm{mW}/\mathrm{mm}}^2$ (red inverted triangles: high yellow). Insets represent examples of averaged fEPSPs before (1) and after (2) LTP induction as indicated by horizontal grey and black bars. Scales: 0.2 mV, 10 ms. Right: Summary data for relative fEPSP slopes averaged during (2) for each slice. $n=14$ slices from 4 mice (Arch-no yellow), $n=17$ slices from 10 mice (Arch-low yellow), $n=5$ slices from 3 mice (Arch-high yellow), $n=16$ slices from 4 mice (no Arch-no yellow), $n=10$ slices from 5 mice (no Arch-low yellow), and $n=8$ slices from 7 mice (no Arch-high yellow). **$p<0.01$, ***$p<0.001$, compared to 1, which is the averaged relative fEPSP slope during the baseline indicated by gray bars (1), Wilcoxon’s signed rank test. Boxes and the thick bars inside represent SEM and means.

LTP induction in the dmPFC-BLA input was tested by giving trains of blue light pulses alone or combined with the continuous yellow light of different intensities [Fig. 4(d)]. The yellow light by itself did not cause the release of glutamate from the dmPFC axonal terminals in BLA (data not shown). Unexpectedly, the trains of blue light in the absence of yellow light induced a significant LTP [Fig. 4(e), left, black-filled circles]. Combining the trains of blue light and the yellow light of low intensity ($0.15{\mathrm{mW}/\mathrm{mm}}^2$) produced more significant LTP and with a tendency to be higher than with the blue light alone [Fig. 4(e), left, orange-filled circles]. Increasing the yellow irradiance to $0.24{\mathrm{mW}/\mathrm{mm}}^2$ prevented LTP induction [Fig. 4(e), left, red-filled inverted triangles]. In slices without Arch, the low-intensity or the high-intensity yellow light did not enable LTP induction by the pulses of blue light [Fig. 4(e), middle]. Together, these data indicate that Arch enables LTP induction by the trains of blue light, and it occurs even in the absence of yellow light, suggesting that the blue light inhibits Sst-INs expressing Arch. Consistently, whole-cell recordings from an Sst-IN with Arch revealed hyperpolarizing currents elicited by blue light ( Supplemental Fig. S2). We also found that continuous yellow light at $0.24{\mathrm{mW}/\mathrm{mm}}^2$ inhibited EPSCs evoked in BLA neurons by stimulating Chronos-expressing dmPFC axons with pulses of blue light ( Supplemental Fig. S3). It may result from the desensitization of Chronos by yellow light and would explain the failure of LTP induction in the presence of a stronger yellow light.

To test LTP induction in vivo, mice expressing Arch in the BLA Sst-INs and Chronos in dmPFC were implanted with optrodes, whose two electrodes were positioned in BLA and an optical fiber above BLA [Figs. 5(a) and 5(b)]. The LTP induction protocol, identical to the protocol used ex vivo, but repeated three times with 1-h interval, produced LTP, which lasted for almost 10 h [Figs. 5(c) and 5(d)].

Fig. 5

Arch-assisted LTP induction in vivo. (a) Left: LED light source-optrode assemblies. Right: a mouse implanted with two optrodes aiming bilaterally at BLA. (b) An example position of electrodes (red asterisks) in a BLA slice imaged under the visible (left) or fluorescent (right) light. The fluorescence arises from Chronos-GFP in dmPFC axons and Arch-GFP in Sst-INs. (c) An example of an LTP experiment showing the slope (red) and amplitude (blue) of light-evoked fEPSP. Light-blue arrows show the trains of 50-Hz light stimulation. The horizontal gray/black bars indicate the ranges for averaging for the sweeps shown on the right (1, gray, 1 h before LTP induction; 2, black, the 3 h after the end of LTP induction). Scales: 0.4 mV, 5 ms. (d) Left: summary LTP experiment. Each data point represents a single independently tested amygdala ($n=6$ amygdalae from three mice). Right: summary data for the relative fEPSP slope and amplitude facilitation during the 3 h after the end of LTP induction, identified by the horizontal black bar (2). *$p<0.05$, compared to 1, which is the averaged relative fEPSP slope/amplitude during the baseline indicated by gray bars (1), Wilcoxon’s signed rank test.